含CpG-ODN基序重组抑制素质粒的构建及其对小鼠颗粒细胞相关凋亡基因表达的影响

doi:10.11843/j.issn.0366-6964.2018.08.007

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (8): 1625-1632.doi: 10.11843/j.issn.0366-6964.2018.08.007

含CpG-ODN基序重组抑制素质粒的构建及其对小鼠颗粒细胞相关凋亡基因表达的影响

阳美霞^1,2, 张虹亮¹, 李蓝祁¹, 张羽芳¹, 杨利国^3*, 王水莲^1,2*

1. 湖南农业大学动物医学院, 长沙 410128;
2. 湖南省兽药工程技术研究中心, 长沙 410128;
3. 华中农业大学动物科技学院, 武汉 430070

收稿日期:2017-12-12 出版日期:2018-08-23 发布日期:2018-08-23
通讯作者: 杨利国,博士,教授,博士生导师,主要从事动物生殖调控研究,E-mail:yangliguo2006@126.com;王水莲,博士,教授,博士生导师,主要从事动物生殖调控研究,E-mail:wangshuilian1234@126.com
作者简介:阳美霞(1994-),女,湖南邵阳人,硕士生,主要从事动物生殖调控研究,E-mail:1217360984@qq.com
基金资助:
国家自然科学基金（31672507）

Construction of Recombinant Inhibin Plasmid Containing CpG-ODN Motif and Its Effect on the Expressions of Apoptosis-related Genes in Mouse Granulosa Cells

YANG Mei-xia^1,2, ZHANG Hong-liang¹, LI Lan-qi¹, ZHANG Yu-fang¹, YANG Li-guo^3*, WANG Shui-lian^1,2*

1. College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China;
2. Hunan Engineering Research Center of Veterinary Drug, Changsha 410128, China;
3. College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

Received:2017-12-12 Online:2018-08-23 Published:2018-08-23

摘要/Abstract

摘要：

旨在构建含免疫基序CpG-ODN片段的重组抑制素质粒pEGISI-CpG-ODN，并研究其对小鼠颗粒细胞中相关凋亡基因mRNA表达水平的影响，以探讨免疫佐剂CpG-ODN对小鼠卵巢颗粒细胞的作用。采集21~23日龄雌性ICR小鼠卵巢颗粒细胞，试验分为3组：空载体组（pEGFP）、抑制素重组质粒组（pEGISI）和含CpG-ODN基序的重组抑制素质粒组（pEGISI-CpG-ODN）。采用普通PCR扩增目的片段CpG-ODN，荧光定量RT-PCR（qRT-PCR）检测转染后细胞中CpG及死亡受体通路相关基因（Fas、FasL、DR4/5、TRAIL、Caspase8和Caspase3）mRNA的表达水平。结果表明：重组抑制素质粒pEGISI-CpG-ODN构建成功；重组抑制素质粒（pEGISI-CpG-ODN）转染颗粒细胞后，CpG基因能在细胞中高表达；与pEGISI组相比，pEGISI-CpG-ODN组转染颗粒细胞后，能极显著降低细胞中促凋亡基因Fas、FasL、DR4/5、TRAIL、Caspase8和Caspase3 mRNA的表达水平（P<0.01）。综上表明，CpG-ODN可拮抗抑制素的作用，下调小鼠颗粒细胞中凋亡基因的表达，进而影响卵泡发育。

Abstract:

The purpose of this study was to construct a recombinant inhibin plasmid containing a CpG-ODN fragment with immunogenicity(named as pEGISI-CpG-ODN), and determine its effect on mRNA expression levels of apoptosis-related genes to investigate the roles of CpG-ODN in mouse ovarian granulosa cells (GCs). GCs were obtained from the ovaries of 21-23 days female ICR mice. The experiment was divided into 3 groups:empty vector group (pEGFP), inhibin recombinant plasmid group (pEGISI) and containing CpG-ODN motif recombinant inhibin plasmid group (pEGISI-CpG-ODN). The target fragment CpG-ODN was amplified by PCR. The mRNA expression levels of CpG and the death receptor pathway related genes (Fas, FasL, DR4/5, TRAIL, Caspase8 and Caspase3) in transfected cells were analyzed by quantitative real-time PCR (qRT-PCR). The results showed that a new recombinant inhibin plasmid called pEGISI-CpG-ODN was constructed successfully. Moreover, CpG gene could be highly expressed in GCs after transfection of GCs with recombinant inhibin plasmid (pEGISI-CpG-ODN). Furthermore, the mRNA expression levels of pro-apoptotic genes Fas, FasL, DR4/5,TRAIL, Caspase8 and Caspase3 were very significantly lower in GCs of the pEGISI-CpG-ODN plasmid treated group than those in pEGISI treated group (P<0.01). These results suggested that CpG-ODN might not only resist the function of inhibin but also decrease the expression levels of apoptotic genes in mouse GCs, as a result, it was able to affect follicular development.

中图分类号:

S854.4

阳美霞, 张虹亮, 李蓝祁, 张羽芳, 杨利国, 王水莲. 含CpG-ODN基序重组抑制素质粒的构建及其对小鼠颗粒细胞相关凋亡基因表达的影响[J]. 畜牧兽医学报, 2018, 49(8): 1625-1632.

YANG Mei-xia, ZHANG Hong-liang, LI Lan-qi, ZHANG Yu-fang, YANG Li-guo, WANG Shui-lian. Construction of Recombinant Inhibin Plasmid Containing CpG-ODN Motif and Its Effect on the Expressions of Apoptosis-related Genes in Mouse Granulosa Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(8): 1625-1632.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2018.08.007

https://www.xmsyxb.com/CN/Y2018/V49/I8/1625

参考文献

[1] GILCHRIST R B.Recent insights into oocyte-follicle cell interactions provide opportunities for the development of new approaches to in vitro maturation[J].Reprod Fertil Dev,2011,23(1):23-31.
[2] KOMATSU K,MASUBUCHI S.Observation of the dynamics of follicular development in the ovary[J].Reprod Med Biol,2017,16(1):21-27.
[3] MATSUDA F,INOUE N,MANABE N,et al.Follicular growth and atresia in mammalian ovaries:Regulation by survival and death of granulosa cells[J].J Reprod Dev,2012,58(1):44-50.
[4] GAO F,ZHANG J,WANG X N,et al.Wt1 functions in ovarian follicle development by regulating granulosa cell differentiation[J].Hum Mol Genet,2014,23(2):333-341.
[5] STENVERS K L,FINDLAY J K.Inhibins:From reproductive hormones to tumor suppressors[J].Trends Endocrinol Metab,2010,21(3):174-180.
[6] BILEZIKJIAN L M,JUSTICE N J,BLACKLER A N,et al.Cell-type specific modulation of pituitary cells by activin,inhibin and follistatin[J].Mol Cell Endocrinol,2012,359(1-2):43-52.
[7] LEI M M,CAI L P,LI H,et al.Transcriptome sequencing analysis of porcine granulosa cells treated with an anti-inhibin antibody[J].Reprod Biol,2017,17(1):79-88.
[8] TU F,PAN Z X,YAO Y,et al.miR-34a targets the inhibin beta B gene,promoting granulosa cell apoptosis in the porcine ovary[J].Genet Mol Res,2014,13(2):2504-2512.
[9] GENG L Y,FANG M,YI J M,et al.Effect of overexpression of inhibin α(1-32) fragment on bovine granulosa cell proliferation,apoptosis,steroidogenesis,and development of co-cultured oocytes[J].Theriogenology,2008,70(1):35-43.
[10] SCHEIERMANN J,KLINMAN D M.Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer[J].Vaccine,2014,32(48):6377-6389.
[11] SUN R,YUE F,QIU L M,et al.The CpG ODNs enriched diets enhance the immuno-protection efficiency and growth rate of Chinese mitten crab,Eriocheir sinensis[J].Fish Shellfish Immunol,2013,35(1):154-160.
[12] URSU R,CARPENTIER A F.Immunotherapeutic approach with oligodeoxynucleotides containing CpG motifs (CpG-ODN) in malignant glioma[C]//YAMANAKA R.Glioma.New York:Springer,2012:95-108.
[13] FLAMINIO M J B F,BORGES A S,NYDAM D V,et al.The effect of CpG-ODN on antigen presenting cells of the foal[J].J Immune Based Ther Vaccines,2007,5:1.
[14] HARTMANN G,WEERATNA R D,BALLAS Z K,et al.Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo[J].J Immunol,2000,164(3):1617-1624.
[15] YEH D W,LAI C Y,LIU Y L,et al.CpG-oligodeoxynucleotides developed for grouper toll-like receptor (TLR) 21s effectively activate mouse and human TLR9s mediated immune responses[J].Sci Rep,2017,7(1):17297.
[16] SU H,LIAO Z W,YUAN G L,et al.A plasmid containing CpG-ODN as vaccine adjuvant against grass carp reovirus in grass carp Ctenopharyngodon idella[J].Oncotarget,2017,8(49):86576-86591.
[17] 曹少先,茆达干,舒邓群,等.卵泡抑制素与GFP融合基因疫苗pEGISI的构建及表达[J].家畜生态学报,2007,28(2):9-12.
CAO S X,MAO D G,SHU D Q,et al.Construction and expression of inhibin DNA vaccine pEGISI in 293T cell[J].Acta Ecologiae Animalis Domastici,2007,28(2):9-12. (in Chinese)
[18] MELDI K M,GACONNET G A,MAYO K E.DNA methylation and histone modifications are associated with repression of the inhibin α promoter in the rat corpus luteum[J].Endocrinology,2012,153(10):4905-4917.
[19] KEMPISTY B,PIOTROWSKA H,RYBSKA M,et al.Expression of INHβA and INHβB proteins in porcine oocytes cultured in vitro is dependent on the follicle size[J].Zygote,2015,23(2):205-211.
[20] HAN L,ZHEN Y H,LIANG A X,et al.Oral vaccination with inhibin DNA delivered using attenuated Salmonella choleraesuis for improving reproductive traits in mice[J].J Basic Microbiol,2014,54(9):962-968.
[21] CAI L P,SUN A D,LI H,et al.Molecular mechanisms of enhancing porcine granulosa cell proliferation and function by treatment in vitro with anti-inhibin alpha subunit antibody[J].Reprod Biol Endocrinol,2015,13:26.
[22] FOSSATI S,GHISO J,ROSTAGNO A.TRAIL death receptors DR4 and DR5 mediate cerebral microvascular endothelial cell apoptosis induced by oligomeric Alzheimer's Aβ[J].Cell Death Dis,2012,3(6):e321.
[23] VAN RAAM B J,LACINA T,LINDEMANN R K,et al.Secretory stressors induce intracellular death receptor accumulation to control apoptosis[J].Cell Death Dis,2017,8(10):e3069.
[24] SANJAYA A,ELDER J R,SHAH D H.Identification of new CpG oligodeoxynucleotide motifs that induce expression of interleukin-1β and nitric oxide in avian macrophages[J].Vet Immunol Immunopathol,2017,192:1-7.
[25] YUAN F,CHU Y,QI L H,et al.Immunoprotection induced by CpG-ODN/Poly(I:C) combined with recombinant gp90 protein in chickens against reticuloendotheliosis virus infection[J].Antiviral Res,2017,147:1-10.
[26] STACEY K J,BLACKWELL J M.Immunostimulatory DNA as an adjuvant in vaccination against Leishmania major[J].Infect Immun,1999,67(8):3719-3726.
[27] MOLDOVEANU Z,LOVE-HOMAN L,HUANG W Q,et al.CpG DNA,a novel immune enhancer for systemic and mucosal immunization with influenza virus[J].Vaccine,1998,16(11-12):1216-1224.
[28] ZHANG W D,AN M,XI J C,et al.Targeting CpG adjuvant to lymph node via dextran conjugate enhances antitumor immunotherapy[J].Bioconjug Chem,2017,28(7):1993-2000.
[29] YU X Q,WANG Y H,LIN J,et al.Lipopolysaccharides-induced suppression of innate-like B cell apoptosis is enhanced by CpG oligodeoxynucleotide and requires toll-like receptors 2 and 4[J].PLoS One,2016,11(11):e0165862.
[30] LU C,HA T Z,WANG X H,et al.The TLR9 ligand,CpG-ODN,induces protection against cerebral ischemia/reperfusion injury via activation of PI3K/Akt signaling[J].J Am Heart Assoc,2014,3(2):e000629.

含CpG-ODN基序重组抑制素质粒的构建及其对小鼠颗粒细胞相关凋亡基因表达的影响

Construction of Recombinant Inhibin Plasmid Containing CpG-ODN Motif and Its Effect on the Expressions of Apoptosis-related Genes in Mouse Granulosa Cells

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	李昕, 孙燕燕, 林密, 陈夏辉, 李峰松, 包艳芳, 杨光, 蒋韬. O、A、Asia 1型口蹄疫病毒胶体金定量检测免疫层析方法的建立[J]. 畜牧兽医学报, 2021, 52(4): 1042-1052.
[2]	张雪婷, 陈峥嵘, 赵雯雯, 韩丽, 陈建国. 区分犬瘟热病毒Asia-Ⅰ型野毒株及疫苗株的AS-PCR方法[J]. 畜牧兽医学报, 2020, 51(10): 2490-2499.
[3]	徐璐, 夏应菊, 张乾义, 张雯雯, 王震, 李翠, 邹兴启, 朱元源, 徐嫄, 王兆, 赵启祖, 王琴. 猪瘟病毒抗体ELISA检测国家级能力验证结果与分析[J]. 畜牧兽医学报, 2020, 51(9): 2250-2256.
[4]	王俊伟, 陈芳洲, 库旭钢, 李畅, 何启盖. 基于PCV3-Cap蛋白间接ELISA检测方法的建立及临床应用[J]. 畜牧兽医学报, 2019, 50(2): 454-460.
[5]	蒋书东, 张兴旺, 方富贵, 周伟娟, 王登峰, 朱晖. 雌性安徽白山羊胸腺形态学增龄性变化[J]. 畜牧兽医学报, 2018, 49(8): 1743-1751.
[6]	朱建波, 杨振兴, 肖雷, 高林, 苗海生, 何于雯, 孟锦昕, 杨恒, 李华春. 流行性出血病多克隆抗体C-ELISA检测方法的建立[J]. 畜牧兽医学报, 2018, 49(7): 1440-1450.
[7]	罗尚星, 范京惠, 刘宝京, 师乾凯, 侯林杉, 左玉柱. 猪丁型冠状病毒与猪流行性腹泻病毒双重实时荧光定量RT-PCR方法的建立和初步应用[J]. 畜牧兽医学报, 2018, 49(4): 852-858.
[8]	魏后军, 范志宇, 王芳, 宋艳华, 胡波, 仇汝龙, 陈萌萌, 徐为中, 薛家宾. 检测兔出血症病毒抗体胶体金试纸条的研制及初步应用[J]. 畜牧兽医学报, 2018, 49(3): 614-619.
[9]	王佳, 熊祺琰, 华利忠, 刘蓓蓓, 甘源, 冯志新, 刘茂军, 武昱孜, 邵国青. 猪鼻支原体血清抗体间接ELISA检测方法的建立[J]. 畜牧兽医学报, 2017, 48(10): 1932-1938.
[10]	马锐，尹人杰，黄小波，文心田，杨国淋，常晓霞，张仙，滑翔，赵玉佳，曹三杰，文翼平，伍锐. 猪病毒性腹泻检测基因芯片的两种样品标记技术比较[J]. 畜牧兽医学报, 2016, 47(4): 752-761.
[11]	滑翔，胡中凯，黄小波，文心田，曹三杰，文翼平，伍锐，邓静，赵松，尹人杰，常晓霞，欧阳达，张仙. 检测猪流行性腹泻病毒、猪传染性胃肠炎病毒和猪轮状病毒的cDNA芯片的构建[J]. 畜牧兽医学报, 2015, 46(12): 2235-2242.
[12]	王晨燕，王隆柏，车勇良，吴学敏，陈如敬，邵良平,周伦江. 应用双向电泳分析2株不同毒力的副猪嗜血杆菌蛋白质表达的差异[J]. 畜牧兽医学报, 2015, 46(3): 503-508.
[13]	张改梅，贾红，侯绍华，鑫婷，郭晓宇，袁维峰，刘淑清，吴竞，高新桃，李明，董瑞凯，朱鸿飞. 检测牛IFN-γ双抗体夹心ELISA的建立及在牛结核病诊断上的初步应用[J]. 畜牧兽医学报, 2014, 45(10): 1693-1698.
[14]	彭武丽，季新成，于学辉，史茜，王科珂，李娜. 多重PCR检测奶牛布鲁菌、鹦鹉热衣原体和贝纳氏柯克斯氏体[J]. 畜牧兽医学报, 2014, 45(1): 123-128.
[15]	王鑫，崔治中，龚建森，高明燕，范建华，俞燕，朱静，徐步. ELISA与IFA用于检测J亚型禽白血病病毒的比较分析[J]. 畜牧兽医学报, 2013, 44(9): 1499-1503.